Castanea mollissima Blume shell was collected from Xindeng Town, Hangzhou City, Zhejiang Province, China, and was dried and grinded for further use. Pleurotus geesteranus spawn and cottonseed hull were supplied by Wuhan SuiSuiFeng Agricultural Technology Development Co., Ltd., China. Rice bran, lime and gypsum were collected from the Fuyang District, Hangzhou, Zhejiang, China as shown in Table 1.

Four cottonseed hull substrates (CK, A, B and C) containing different contents of Castanea mollissima Blume shell were prepared, among which the pure cottonseed hull substrate (CK) was used as the control, as shown in Table 2.

The ingredients of each substrate were weighted and homogenously blended. The water content of each substrate was adjusted to 55% - 60% with water. The substrate was mixed well and bagged into polypropylene plastic bags (17 cm × 33 cm × 0.5 cm). The number of bags and the dry weight of each bag were recorded. The bagged substrates were autoclaved for sterilization at 121˚C under 103.4 KPa for 2 h, cooled to room temperature, inoculated and cultured in a dark culture chamber at 20˚C - 23˚C and the humidity of 50% - 70%. The collar and lid were removed after the mycelia completely covered the bag. The inoculated substrates were then moved to the mushroom cultivation room of the temperature of 22˚C - 24˚C and the humidity of above 90%. The cultivation room was consistently ventilated to avoid the accumulation of carbon dioxide that might cause the malformation of Pleurotus geesteranus. The Pleurotus geesteranus were harvest as the mushroom caps became 2 - 3 cm big.

For each substrate, 10 bags were randomly selected for the measurement of mycelial growth rate. The lengths of mycelia were measured every 5 days and the mycelial growth rate at the measuring time was calculated. The last measurement was conducted when the mycelia completely covered the bag. The average growth rate on each substrate was then calculated and analyzed [11] .

-, not detected.

A-C, Castanea mollissima Blume shell containing substrate; CK, conventional cottonseed hull substrate.

Biological efficiency (%) = yield per bag (g)/dry weight per bag (g) × 100%.

Fresh Pleurotus geesteranus were harvested and the residues of stipe were removed. The mushrooms were dried in an oven at 60˚C and pulverized for the measurements. The contents of protein, fat, crude fiber and amino acids were determined according to the protocol of GB 5009.5-2016, GB 5009.6-2016, GB/T 5009.10-2003 and GB 5009.124-2016 respectively. The contents of heavy metals including mercury (Hg), cadmium (Cd), arsenic (As) and lead (Pb) were determined according to the method of GB 5009.268-2016. Each measurement was repeated three times and the mean value was reported.

All data analyses and processing were conducted in EXCEL.

Figure 1 shows the mycelial growth rates of Pleurotus geesteranus on substrates CK and A-C. It is clear that the mycelial growth rates on the substrates containing Castanea mollissima Blume shell are higher than that on substrate CK, following the order of A > B > C > CK. The mycelial growth rate on substrate A containing 10% Castanea mollissima Blume shell is the highest 0.49 ± 0.025 cm/d,

0.15 ± 0.005 cm/d higher than that on the control substrate CK. Both of the contents of tannin and saponin, as well as C/N, increase with the increase of the shell content in substrate, which causes the decrease of mycelial growth rate. These results indicate that partially substituting cottonseed hull substrate with Castanea mollissima Blume shell can accelerate the growth of Pleurotus geesteranus. However, the mycelial growth rate of Pleurotus geesteranus decreases with the increase of the shell content. The suitable C/N of substrate for the cultivation of Pleurotus geesteranus is 27. Higher or lower C/N ratios slow the mycelial growth of Pleurotus geesteranus.

The Pleurotus geesteranus cultivated on the 4 substrates exhibited significantly different yields and biological efficiencies (Table 3) with the order of B > C > CK > A. Therefore, both of the yield and biological efficiency of Pleurotus geesteranus increase first and then decrease with the increase of the shell content. The highest yield and biological efficiency were obtained on substrate B containing 30% Castanea mollissima Blume shell with the C/N of 29.54. Increasing the Castanea mollissima Blume shell content of substrate increases the C/N ratio and decreases the yield and biological efficiency of the cultivated Pleurotus geesteranus.

The protein in edible fungi is considered as the “plant meat” and is a very good source of protein. In general, edible fungi contain usually 15% - 60% proteins [13] . The protein content of Pleurotus geesteranus cultivated on the Castanea mollissima Blume shell containing substrate increased with the increases of the shell content and the C/N ratio of substrate (Figure 2). The protein content obtained on substrate C reached up to 28.3 ± 0.83 g/100 g, 4.5 ± 0.08 g/100 g higher than that obtained on substrate CK. Therefore, it can be concluded that the protein content of Pleurotus geesteranus can be improved by blending certain amounts of Castanea mollissima Blume shell to the substrate and is increased with the increase of the C/N of substrate.

Note: 385 g dry substrate per bag.

The fat contents of edible fungi are low, usually lower than 10% [13] . The Pleurotus geesteranus cultivated on the substrates exhibited the fat contents in the order of CK > A > B > C (Figure 3), among which, the fat content of substrate C was lower 0.8 ± 0.03 g/100g, which was 1.1 ± 0.04 g/100g lower than that of group CK. Suggesting that adding Castanea mollissima Blume shell substrate could reduce the fat content of the mushroom.

Crude fiber refers to the insoluble dietary fibers mainly including cellulose, hemicellulose and lignin. As shown in Figure 4, the crude fiber content of Pleurotus geesteranus increased with the increases of the shell content and the C/N of substrate. The crude fiber content reached up to 10.63% ± 0.89% on substrate B containing 30% shell, which was 8.43% ± 0.71% higher than that obtained on substrate CK. Further increasing the shell content to 50% decreased the crude fiber content, yet the crude fiber content was still 6.92% ± 0.59% higher than that obtained on substrate CK. These results suggest that a certain amount of Castanea mollissima Blume shell in cottonseed hull substrate can increase the crude fiber content of the cultivated Pleurotus geesteranus.

Edible fungi contain 17 - 18 of the 20 amino acids required by the human body, and can provide almost all 8 essential amino acids, especially lysine, methionine and threonine that are lacked in cereals [13] . Table 4 lists the contents of amino acids, the total content of amino acids, the total content of essential amino acids, the total content of non-essential amino acids and the total content of umami amino acids of the Pleurotus geesteranus cultivated on different substrates. It is

*essential amino acid; ^#umami amino acid.

clear all of them are in the order of C > B > A > CK. In addition, the total essential amino acids account for over 37.78% of the total amino acids and are more than 60% of the total non-essential amino acids in the Pleurotus geesteranus cultivated on the Castanea mollissima Blume shell containing substrates. In contrast, in the Pleurotus geesteranus cultivated on substrate CK, the total essential amino acid content accounts for 31.59% of the total amino acid content and is only 46.19% of the total non-essential amino acid content. In all, the total contents of all kinds of amino acids increased with the increases of the shell content and the C/N of substrate. Therefore, Castanea mollissima Blume shell can be used as a substrate material to increase the total amino acid content and essential amino acid content of Pleurotus geesteranus.

The national standard of China GB 7096-2003 Hygienic standard for edible fungi requires total arsenic (As) ≤ 1.0 mg/kg, total mercury (Hg) ≤ 0.2 mg/kg and total lead (pb) ≤ 2.0 mg/kg in edible fungi. China National Food Safety Standard: Limits of Contaminants in Foods GB 2762-2012 stipulates that the Pb in foods is no more than 1.0 mg/kg and that of Cd is no more than 0.5 mg/kg. Green Food-Edible FungiGB 2762-2012 NY 749-2012 states the limits of Pb and Cd of no more than 1.0 mg/kg and 0.5 mg/kg, respectively. All contents of Hg, As, Cd and Pb of Pleurotus geesteranus increased with the increase of the Castanea mollissima Blume shell content in substrate and the Cd content became higher than the limit as the shell content increased to 50%. Therefore, Castanea mollissima Blume shell content in substrate for the cultivation of Pleurotus geesteranus should be no more than 30% (Table 5).